The present invention provides a method for enhancing the immunogenicity of weakly immunogenic or non-immunogenic cells in order to provide the immune system with an immunogenic signal capable of stimulating T cell activation leading to an effective immune response. The method of the invention generates cellular vaccines which are useful for the prevention and treatment of diseases which develop and/or persist by escaping the immune response triggered by T cell activation. Such diseases include, for example, all cancers, natural and induced immune-deficiency states, and diseases caused by infections with a variety of pathogens.
U.S. Pat. No. 5,484,596 by Hanna et al. describes using tumor tissue as a vaccine. U.S. Pat. No. 4,844,893 by Honsik et al. describes arming IL-2-activated leukocytes with Mabs directed to antigens preferentially expressed on tumor cells for killing the target cells. Both patents are incorporated by reference herein.
Anti-tumor immune responses are mediated primarily by T lymphocytes. Down regulation of both the major histocompatibility complex (MHC) and the molecules that costimulate the immune response is associated with defective T cells activation signaling by tumor cells (Luboldt et al., Cancer Res. 56:826-830, 1996; L. Chen et al., 1992, Cell 1: 1093; P. S. Linsley, J. A. Ledbetter, 1993, Ann. Rev. Immunol 11: 191; G. J. Freeman et al., 1993, Science 262: 909; C. H. June et al., 1994, Immune. Today 15: 321; J. T. Gerge et al., 1993, Cancer Res. 53: 2374; Ostrand-Rosenberg, 1993, S. Curr. Opin. Immune. 6: 772; B. E. Elliot et al., 1989, Adv. Cancer Res. 53: 181).
T cell receptor (TCR) recognition of MHC-bound antigen is not a sufficient signal for T cell activation. Costimualtory molecules, such as B7-1 and B7-2, are cell surface proteins of antigen presenting cells (APCs), and other cells targeted by the immune response, that provide critical signals for T cell activation (for review, see L. Chen et al., 1995, Immunol. Rev. 145: 123; T. Tykocinski et al., 1996, Am. J. Path. 148: 1). B7 signaling via the T cell surface molecule CD28 appears to be the major costimulatory pathway for T cell activation. However, recent studies show that costimulation is a more complex event which involves both cytokines and adhesion molecules (G. Yang et al., 1995, J. Immune. 154: 2794; M. Kubin et al., 1994, J. Exp. Med. 180: 211; Y. Li et al., 1996, J. Exp. Med. 183: 639).
Many approaches have been used to enhance the immunogenicity of tumor cells (see, for example, the references cited in this section). The major approaches presently under investigation involve gene transfer. In this regard, most of the methods employed to date have required ex vivo or in vivo transfection with genes such as MHC or B7, or modification of tumor cells with antigen presenting cells (APCs) (Y. J. Guo et al., 1994, Science 263: 518; M. Tykocinski, 1996, A. J. Path. 148: 1; J. Young and K. Inaba, 1996, J. Exp. Med. 183: 7; L. Zitvogel et al., 1996, J. Exp. Med. 183: 87; C. M. Celluzzi et al., 1996, J. Exp. Med. 183: 283). These approaches are time consuming and problematic because of the poor transfectability of primary tumor cells and because of the requirement for large numbers of APCs.
In vitro treatment of tumor cells with cytokines increases the expression of MHC and adhesion molecules (R. Mattsson et al., 1992, Biol-Reprod 46: 1176; R. J. Ulevitch et al., 1991, Am. J. Pathol. 139: 287; F. Willems et al., 1994, Eur. J. Immune. 24: 1007; I. Saito et al., 1993, J. Clin. Lab. Anal. 7: 180; R. A. Panettieri et al., 1994, J. Immune. 154: 1358; M. Ikeda et al., 1994, J. Invest. Dermatol. 103: 791). Transfection of tumor cells with MHC, B7-1 and B7-2 genes converts low immunogenic tumor cell lines to immunogenic cell lines (S. E. Townsend and J. P. Allison, Science 259: 368; J. P. Allison et al., 1995, Curr. Opin. Immune. 7: 682; G. Yang et al., 1995, J. Immune. 154: 2794; M. Kubin et al., 1994, J. Exp. Med. 180: 211). Non-immunogenic tumor cells are not responsive to transfection with the B7 gene alone but can become responsive by co-expression of CD48 molecules at the cell surface (Y. Li et al., 1996, J. Exp. Med. 183: 639).
The costimulatory molecule B7 can under some circumstances deliver a negative signal through its binding to CTLA-4, a second receptor for B7 on T cells. Cross-linking CTLA-4 molecules in vitro has been shown to inhibit T cell proliferation. Furthermore, mice deficient in CTLA-4 develop severe T cell proliferative disorders (K. Kawai et al., 1993, Science 261: 609; J. P. Allison, M. K. Krummel, 1995, Science 270: 932; J. M. Green et al., 1994, Immunity 1: 501). A recent report showed that the introduction of anti-CTLA-4 monoclonal antibody (MAb), which blocks CTLA-4 mediated signaling, resulted in enhanced T cell-dependent rejection of tumors in certain mouse models (D. R. Leach et al., 1996, Science 271: 1734). These data provide evidence that CTLA-4 may be counter-regulatory to the CD28 costimulatory signal. Thus, transfected tumor cells expressing B7 molecules may fail to elicit effective immunity due to CTLA-4 mediated negative signaling.
In addition to T cell activation using B7 gene transfection, bispecific monoclonal antibodies (Bi-MAbs) in combination with pre-stimulated lymphocytes have been used to induce T cell activation under certain circumstances. For example, one study reports that costimulatory signals can be delivered by a combination of Bi-MAbs to CD28:CD30 (CD30 is a Hodgkin""s tumor-associated antigen) and CD3:CD30 in combination with peripheral blood lymphocytes (PBLs) pre-stimulated with the CD3:CD30 Bi-MAb in the presence of CD30+ Hodgkin""s tumor-derived cells; however, the combination of CD28:CD30 and CD3:CD30 Bi-MAbs alone did not induce significant in vitro cytotoxicity of resting human PBLs against a Hodgkin""s tumor-derived cell line, and stimulation with the CD28:CD30 Bi-MAb alone was not effective (C. Renner et al., 1994, Science 264: 833). Similarly, regression of Hodgkin""s derived tumor xenografts was observed only when both the CD28:CD30 and CD3:CD30 Bi-MAbs were used in combination with PBLs prestimulated in vitro with CD30+ cells and CD3:CD30 Bi-MAb; no significant effect was observed in xenografts treated with either of the Bi-MAbs alone, or a combination of the two Bi-MAbs without prestimulated human PBLs (Renner et al., supra).
The present invention features immunogenic tumor cells and other immunogenic autologous cells, convenient methods of making such immunogenic cells, methods of using such immunogenic cells to activate or enhance immune response against diseased cells with minimum effect on normal or healthy cells, and methods of avoiding the negative T cell signaling pathway.
The present invention provides a method for enhancing the immunogenicity of weakly-immunogenic or non-immunogenic cells, resulting in a cellular vaccine that can stimulate T cell activation, which in turn leads to an effective immune response against diseased cells. The cellular vaccines of the present invention can be used as vaccines to prevent diseases and as immunotherapeutics to treat diseases. The starting materials for the cellular vaccine can be a target diseased cell (e.g., autologous or in vivo diseased cells and in vitro transformed cell lines), or an antigen presenting cell presenting one or more antigens associated with a disease (e.g., dendritic cells, macrophages, B cells, and other cells fused with diseased cell, pulsed with antigens or transfected with antigen expressing nucleic acid).
In summary, the method of the invention involves the steps of (1) treating weakly- or non-immunogenic autologous cells (target cells) in order to amplify primary and costimulatory T cell activation signals in the cells, and (2) attaching to the treated cells a substance capable of binding to one or more antigens on the treated cells and to one or more T cell activation costimulatory molecules on the surface of T cells (such as CD28), thereby providing the treated cells with the capacity to physically link to T cells and to activate the costimulatory signal. Such substances include, but are not limited to, bispecific monoclonal antibodies (Bi-MAbs) targeted to antigen on the treated cells and to CD28 and/or other costimulatory molecules on T cells. The first step may be skipped when the autologous cell is attached with (1) a bridge molecule with two or more binding sites for T cell activation costimulatory molecules on the surface of T cells, or (2) two or more bridge molecules each with one or more binding sites for T cell activation costimulatory molecules on the surface of T cells. The fist step may also be skipped when the target cells are antigen presenting cells presenting one or more antigens associated with a disease.
Once the primary and/or costimulatory T cell activation signals in the target diseased cells have been amplified by cytokines or other means and the bridge molecules have been attached to the target diseased cells, the cytokines and the bridge molecules not attached to the target diseased cells may be removed from the immunogenic composition before the target diseased cells are administered to a patient. This additional step minimizes adverse effects associated with administering cytokines to a patient. It also minimizes the risk associated with allowing bridge molecules not attached to a target diseased cell into a patient, an event which may cause unwanted immune response against normal or healthy cells.
The first step of the method up-regulates antigen processing capacity within the treated cells and amplifies the expression of cell surface molecules involved in T cell activation. The second step provides the treated cells with a means to physically bridge to T cells via CD28 and/or other costimulatory molecules, thereby providing optimal conditions for stimulating T cell activation.
Thus, in a first aspect, this invention features an immunogenic composition for administration to a patient mammal (including a human) having target diseased cells. The immunogenic composition contains an autologous target diseased cell which differs from the diseased cells in the patient in that it processes and presents antigens characteristic of the diseased cells more effectively. For example, the autologous target diseased cell expresses one or more primary (e.g., MHC) and/or costimulatory (e.g., B7-1 and B7-2) T cell activation molecules at a higher level (e.g., 50% higher, preferably 2 folds higher, more preferably 10 folds higher). As described below, there are different ways of enhancing the expression level of the primary and/or costimulatory T cell activation molecules.
In addition, the autologous target diseased cell has attached thereto one or more bridge molecules. Each bridge molecule has one or more binding sites for one or more costimulatory molecules on the surface of effector cells, which include, but are not limited to, T cells, NK cells, macrophages, LAK cells, B cells, and other white blood cells. Preferably, though not required, the bridge molecules have one or more binding sites for one or more antigens on the surface of the target diseased cell and are attached to the target diseased cells at the cell surface antigens. In another preferred embodiment, substantially all (e.g.,  greater than 80%, preferably  greater than 90%, more preferably  greater than 95%) the bridge molecules in the immunogenic composition are attached to the autologous target diseased cells so that the composition is substantially free of bridge molecules not attached to a target diseased cell. In a further preferred embodiment, the immunogenic composition contains a pharmaceutically effective amount of the target diseased cells with bridge molecules attached thereto.
By xe2x80x9cimmunogenicxe2x80x9d is meant the ability to activate the response of the whole or part of the immune system of a mammal, especially the response of T cells.
By xe2x80x9cautologousxe2x80x9d is meant that the target diseased cell is from the patient mammal, or from another patient having a common major histocompatibility phenotype. An autologous target cell may be obtained from the patient mammal or another source sharing the same MHC with methods known to those skilled in the art. Once taken from a patient, an autologous cell may be modified, transfected, and treated by methods described herein.
By xe2x80x9ctarget diseased cellxe2x80x9d is meant a cell causing, propagating, aggravating or contributing to a disease in a patient mammal. Target diseased cells include, but are not limited to, tumor cells (including unmodified tumor cells, tumor cells modified with different approaches, and primary culture). The sources of tumor cells include, but are not limited to, liver cancer, hepatocellular carcinoma, lung cancer, gastric cancer, colorectal carcinoma, renal carcinoma, head and neck cancers, sarcoma, lymphoma, leukemia, brain tumors, osterosarcoma, bladder carcinoma, myloma, melanoma, breast cancer, prostate cancer, ovarian cancer, and pancreas carcinoma.
Target diseased cells may also be cells infected with prions (which cause Mad Cow diseases among others), viruses, bacteria, fungi, protozoa or other parasites (e.g. worms).
Viruses include those described or referred to in Fields Virology Second Edition, 1990, Raven Press, New York, incorporated by reference herein. Examples include, but are not limited to, herpes virus, rhinoviruses, hepatitis virus (type A, B, C and D), HIV, EBV, HPV, and HLV.
Bacteria include those described or referred to in Bergey""s Manual of Determinative Bacteriology Ninth Edition, 1994, Williams and Wilkins, incorporated by reference herein. Examples include, but are not limited to, gram positive and negative bacteria, streptococci, pseudomonas and enterococci, Mycobacterium tuberculosis, Aeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Bacillus sphaericus, Pseudomonas fluorescens, Pseudomonas putida, Serratia liquefaciens, Lactococcus lactis, Xanthomonas maltophilia, Staphylococcus simulans, Staphylococcus hominis, Streptococcus constellatus, Streptococcus anginosus, Escherichia coli, Staphylococcus aureus, Mycobacterium fortuitum, and Klebsiella pneumonia. 
Primary T cell activation molecules include MHC class I, MHC class II and other molecules associated with antigen processing and/or presentation. Costimulatory T cell activation molecules include ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-2, VLA-1, VCAM-1, 4-1-BB, B7-1, B7-2, and other cell adhesion proteins and other cell surface proteins which can activate T cell costimulatory pathways through T cell surface proteins.
By xe2x80x9cbridge moleculexe2x80x9d is meant a molecule or substance which can bring two or more cells together by attaching to the cells with its binding sites. Preferably, a bridge molecule can bring an autologous target diseased cell together with an effector cell and deliver a signal to the effector cell to activate or enhance the effector cell""s immune response against the target. A bridge molecule has one or more binding sites for stimulatory and/or costimulatory molecules on the effector cells. These binding sites can be designed to activate a positive regulator of T cell activation (e.g., CD28, 4-1BB) but avoid stimulating a negative regulator of T cell activation (e.g., CTLA-4). The binding sites can also be designed to blockade a negative regulator of T cell activation (see Leach et al., Science 271:1734-1736, 1996). A bridge molecule may also have one or more binding sites for antigens on the surface of the target diseased cell. Bridge molecules include, but are not limited to, bispecific monoclonal antibodies, fusion proteins, organic polymers, and hybrids of chemical and biochemical materials. The antibodies described or disclosed in U.S. Pat. Nos. 5,601,819, 5,637,481, 5,635,602, 5,635,600, 5,591,828, 5,292,668 and 5,582,996 are incorporated by reference herein.
The antigen on the target cell serving as an anchor for the bridge molecule need not be unique to the target cell when the bridge molecule is attached to the target cell in vitro. Any molecule on the target cell surface can be used to anchor the bridge molecule, including, but not limited to, proteins, glycoproteins, lipids, glycolipids, phospholipids, lipid aggregates, steroids, and carbohydrate groups such as disaccharides, oligosaccharides and polysaccharides (see xe2x80x9cMolecular Biology of The Cell,xe2x80x9d pp47-58, pp276-337, Second Edition, published by Garland Publishing, Inc. NY and London). Examples include transferrin receptor, Low Density Lipoprotein (LDL) receptor, gp55, gp95, gpl 15, gp210, CD44, ICAM-1, ICAM-2, collagen and fibronectin receptor, transferrin receptors, Fc receptor, and cytokine receptors.
Costimulatory molecules on the surface of effector cells may be antigens, fatty acids, lipids, steroids and sugars that can stimulate or costimulate these effector cells"" functions to destroy the target cells. Costimulatory molecules include, but are not limited to, CD1 a, CD1b, CD1c, CD2, CD2R, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD51/61 complex, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CDw65, CD66a, CD66b, CD66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CDw90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CDw101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD110-CD114, CD115, CDw116, CD117, CD118*, CD119, CD120a, CD120b, CDw121a, CDw121b, CD122, CD123*, CDw124, CD125*, CD126, CDw127, CDw128, CD129, CDw130, LFA-1, LFA-2, LFA-3, VLA-1, VCAM-1, VCAM-2, 4-1BB, cytokine and chemokin receptors. In a preferred embodiment, the bridge molecule has a binding site for CD28 or 4-1BB on the surface of T cells.
By xe2x80x9cpharmaceutically effectivexe2x80x9d is meant the ability to cure, reduce or prevent one or more clinical symptoms caused by or associated with the diseased cells in the patient mammal, including, but not limited to, uncontrolled cell proliferation, bacteria infection, and virus infection.
The immunogenic composition may be isolated, enriched or purified for administration to a patient.
By xe2x80x9cisolatedxe2x80x9d in reference to the immunogenic composition is meant that the autologous target diseased cell is isolated from a natural source. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that one or more naturally occurring materials have been removed from the normal environment. Thus, the target diseased cell may be placed in a different cellular environment or in a solution free of other cells. The term does not imply that the target diseased cell is the only cell present, but does indicate that it is the predominate cell present (at least 20-50% more than any other cells) and is essentially free (about 90% pure at least) of other tissues naturally associated with it in the body of the patient. In a preferred embodiment, the composition is substantially free of effector cells such as T cells. In another preferred embodiment, the composition is substantially free of bridge molecules not attached to a target diseased cell. In a third preferred embodiment, the composition is substantially free of cytokines outside of the target diseased cell.
By xe2x80x9cenrichedxe2x80x9d in reference to the immunogenic composition is meant that the autologous target diseased cell constitutes a significantly higher fraction (2-5 fold) of the total cells in the composition than in the diseased tissue in the patient""s body. This could be caused by a person by preferential reduction in the amount of other cells present, or by a preferential increase in the amount of the specific target diseased cells, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other cells present, just that the relative amount of the cell of interest has been significantly increased in a useful manner. The term xe2x80x9csignificantlyxe2x80x9d here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other cells of about at least 2 fold, more preferably at least 5 to 10 fold or even more.
By xe2x80x9cpurifiedxe2x80x9d in reference to the immunogenic composition does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the target diseased cell is relatively purer than in the natural environment. The target diseased cells could be obtained directly from the patient or from cell culture, with or without modifications. Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. In a preferred embodiment, the composition is substantially free of effector cells such as T cells.
The immunogenic composition may contain a pharmaceutically suitable carrier or excipient. Techniques for formulation and administration may be found in Remington""s Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990). The immunogenic composition may be administered to a patient systemically, e.g., by intravenous infusion or subcutaneous injection. A composition of the invention may be administered as a unit dose to a patient mammal, each unit containing a predetermined quantity (e.g., about 1xc3x97105 to about 1xc3x971010, preferably about 1xc3x97106 to about 1xc3x97109, and more preferably about 1xc3x97107 to about 1xc3x97108) of armed and/or activated autologous target diseased cells calculated to produce the desired therapeutic effect in association with the physiologically tolerable aqueous medium as diluent.
The expression of primary and costimulatory T cell activation molecules may be enhanced by various means, for example, in vitro, ex vivo or in vivo treatment of target cells with cytokines or other factors capable of inducing the desired amplification; and in vitro and in vivo transfer to the target cells of MHC genes, adhesion molecule genes, cytokine genes, and/or their respective transcription activators or enhancers. Cytokines include those described or referred to in The Cytokine Handbook, Thomson, A., (ed.), 1994, Academic Press, San Diego, incorporated by reference herein. Examples include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, G-CSF, GM-CSF, inteferons (e.g., IFN xcex1, xcex2, and xcex3), tumor necrosis factors (e.g., INFxcex1, and xcex2) and other chemokines and lymphokines. In a preferred embodiment, IFNxcex3 and TNFxcex1 are used either alone or in combination to enhance the expression of primary and costimulatory T cell activation molecules in autologous target diseased cells.
The bridge molecule may be attached to the target cells by various means, for example, in vitro, ex vivo or in vivo treatment of target cells with the bridge molecule. When a target diseased cell coated with bridge molecules is administered into a patient, it will bind to costimulatory molecules on the surface of the effector cells. The more densely the target diseased cell is coated with bridge molecules, the more effector cells it will be able to bind. In addition, the more binding sites a bridge molecule has for the costimulatory molecules, the more effector cells it will be able to bind.
In that regard, Applicant has found that a cellular vaccine may be prepared without the need of cytokine treatment (to increase the levels of primary and costimulatory T cell activation molecules) when a plurality of bridge molecules are attached to a target cell with binding sites for two or more different costimulatory molecules on the surface of T cells (e.g., CD3, CD28, and 4-1BB). Individual bridge molecules may be attached to different anchor molecules on the surface of the target diseased cell. An individual bridge molecule may also have two or more binding sites for two or more different costimulatory molecules on the surface of T cells.
Thus, in a second aspect, this invention features an immunogenic composition containing an autologous target diseased cell having attached thereto (a) a bridge molecule which has two or more binding sites for two or more different effector cells, (b) a bridge molecule which has two or more binding sites for two or more different costimulatory molecules on the surface of effector cells, (c) two or more bridge molecules each containing a binding site for a different effector cell, (d) two or more bridge molecules each containing a binding site for a different costimulatory molecule on the surface of effector cells, (d) two or more bridge molecules each attached to a different antigen on the target cells, or (e) a combination of two or more of the above.
A pharmaceutically effective amount of an immunogenic composition of this invention may be complemented by a pharmaceutically acceptable carrier before administration to a patient mammal.
Alternatively, a patient may be administered with a pharmaceutical composition containing (1) a pharmaceutically effective amount of a cytokine capable of increasing the level of one or more primary and costimulatory T cell activation molecules in tumor cells, (2) a pharmaceutically effective amount of a bridge molecule containing a binding site for an antigen on the surface of the tumor cells and a binding site for a costimulatory molecules on the surface of T cells, and (3) a pharmaceutically acceptable carrier.
In treating a patient, the autologous target cell may be treated with cytokines or other means of increasing primary and costimulatory T cell activation molecules in vitro before the target cell is administered to the patient. Alternatively, the cytokines may be administered to the patient to increase primary and costimulatory T cell activation molecules in vivo.
In a third aspect, this invention features a method of generating cytotoxic leukocytes against diseased cells in a patient mammal by contacting a population of effector cells (e.g., white blood cells) in vitro with immunogenic compositions described above for a time period sufficient to react with the immunogenic compositions and collecting the treated effector cell population. The cytotoxic leukocytes so generated can then be administered to a patient to treat or prevent diseases. This adoptive immunotherapy can be used alone or in combination with vaccination to treat or prevent diseases.
The method of the invention is useful for the prevention and treatment of diseases which develop and/or persist by escaping immune responses triggered by T cell activation. Such diseases include, for example, all cancers, natural and induced immune deficiency states, and diseases caused by infections with a variety of pathogens. The method of the invention is illustrated herein by demonstrating its application to three different types of human cancers. Cancer cells are by nature generally weakly immunogenic, fail to trigger an effective T cell response, and survive and grow as a result. As demonstrated herein, cancers can be prevented, and established cancers may be cured, by stimulating an effective T cell response using autologous tumor cell vaccines of the invention.
Other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.